Human Genome Project (HGP)

Introduction to the Human Genome Project

• The human genome is the DNA collection in the nucleus of human cells
• It contains 23 pairs of chromosomes
• Serves as identification marks or blueprints
• A genome is an organism's complete set of DNA
• Virtually every cell in the body has a copy of approximately 3 billion DNA base pairs
• The Human Genome Project (HGP) was an international collaboration among scientists
• A joint initiative by many countries to sequence human chromosomes and genes completely, unraveling human genetic information

History of HGP

• Renato Dulbecco advocated for HGP in a 1984 article
• Reasoning was that knowing the human genome sequence would facilitate understanding cancer
• In May 1985, a meeting about the HGP, with Robert Sinsheimer, assembled twelve experts to debate the potential projects
• The US Department of Energy (DOE) pushed for the HGP
• Suggested that knowing the genome sequence would help understand the radiation effects
• HGP started in 1990 after about 5 years of planning
• Done against a background of skepticism as a multinational project
• Involving 20 research institutions mostly in China, France, Germany, Great Britain, Japan, and the US
• Goals were to determine the entire sequence and develop the genetic and physical maps of the entire human genome
• A National Academy of Science Committee report endorsed the project in 1988
• The HGP lasted from 1990–2004
• Craig Venter and Francis Collins announced the complete draft of the human genome sequence in 2001
• They finished the project by 2003 in which 98% of the genome sequenced with >99.9% accuracy
• The first draft of the sequence was released in June 2000
• The final draft released in April 2003, two years ahead of the projected 2005 date

Objectives of HGP

• Unravel the entire human DNA sequence and make the data accessible to the public and scientific community
• Develop efficient technology to sequence human DNA
• Identify variations in SNPs that predispose people to different diseases
• Understand how specific genes/groups of genes interact to determine predisposition of individuals to disease or good health
• Decipher the genetic code of model organisms to facilitate the understanding of gene functions in a more complex genome like that of humans
• Identify the ethical, legal, and social implications of genome research and obstacles that need overcoming
• Developing Bioinformatics tools to facilitate the gathering, analysis, and storage of increasing amounts of sequence data

Modalities of HGP

• Involves gene (DNA) sequencing
• Essential for sequencing the entire gene (genome) of a human being
• Sanger and Maxam-Gilbert sequencing were the most common technologies until a new sequencing era
• Roche's 454 technology appeared first in 2005
• These technologies were commercialized and capable of producing sequences with high throughput and lower cost
• New technologies: "Next Generation Sequencing (NGS) or"High Throughput Sequencing Technologies
• Includes illumina/solexa, Ion torrent sequencing, ABI/SOLID and Pacific Biosciences SMRT

Sanger Sequencing Technology

• Also known as chain termination, dideoxynucleotide, and sequencing by synthesis method
• Uses one strand of double-stranded DNA as a template
• Sequencing is done using chemically modified nucleotides called dideoxy-nucleotides (dNTPs)
• Used for elongation of nucleotide
• Incorporated into the DNA strand, preventing elongation and completion
• Widely used in single or low-throughput sequencing, however, it is difficult to improve the speed
• Limitations prevent the sequencing of complex genomes, expensive, and time-consuming

Maxam-Gilbert Sequencing

• A first-generation sequencing referred to as the chemical degradation method
• Chemical treatment generates breaks proportion of one or two of the four nucleotide bases in each of the four reactions (C, T+C, G, A+G)
• Generates a series of marked fragments that are separated by their size via electrophoresis
• Sequencing is performed without DNA cloning
• Uses toxic and radioactive chemicals

Roche/454 PyroSequencing

• DNA samples are randomly fragmented and attached to a bead
• Bead surfaces uses primers that have oligonucleotides complementary to the DNA fragments
• Each bead is isolated and amplified using PCR emulsion, which produces about one million copies of each DNA fragment on the bead surface
• Beads are transferred to a plate with wells called the picotiter plate (PTP)
• Pyrosequencing technique used activating a series of downstream reactions producing light at the incorporation of a nucleotide
• Detects light emission after incorporating a nucleotide
• Determines the DNA fragment sequence
• Latest instrument launched by Roche/454 is called GS FLX+
• Generates reads with lengths of up to 1000 bp
• Produces ~1 Million reads per run
• Generates relatively long reads with lengths of up to 1000 bp
• Produces ~1 Million reads per run
• Generates relatively long reads easier to map to a reference genome

Oxford Nanopore Sequencing Technology

• It can determine the order of nucleotides in a DNA sequence
• In 2014, Oxford Nanopore Technologies released the MinION device
• Promises to generate longer reads that ensure better resolution, structural genomic variants, and repeat content
• Mobile single-molecule Nanopore sequencing measures four inches in length
• Released for testing by a community as part of the MinION Access Program (MAP)
• The first DNA molecule strand is linked
• Utilizes a hairpin to the complementary strand and the DNA fragment is passed through a protein nanopore
• A nanopore is a nanoscale hole made of proteins or synthetic materials

Challenges of HGP

• Technical reasons relating to eukaryotic islands embedded in heterochromatic repeats, variations and typically high or low GC content
• There exists of highly conserved regions in the human genome whose function have not yet been identified
• The cost for gene sequencing was very high

Ethical, Legal and Social Issues

• Ethical, Legal, and Social Implications (ELSI) program was founded in 1990
• Focused on the possible consequences of genomic research in areas of privacy, genetic technologies, and ethics
• Focused on education of healthcare professionals, policy makers, students, and the public surrounding genetics

Achievements of HGP

• Drawing out the human chromosome map framework
• A large number of genes have been used for medical genetic testing
• Publication of risk factors for adult chronic diseases such as cancer, diabetes, and infectious, autoimmune diseases
• New knowledge leading to a Combined DNA Index System (CODIS) useful in the forensic field
• Introduced a new space in the field of anthropology to identify the origins of humans
• Has led to disease/new drug discovery, genetic testing, and revolutionizing medical practices

Contributions of HGP

• Advances in epidemiology (human genome epidemiology)
• Advances in drug development including toxicology and efficacy research
• Advances in personalized medicine particularly for multifactorial disorders
• Advances in disease discovery and diagnosis
• Advances in biology and technology, developmental biology, neurobiology, and proteomics

Epidemiology from HGP

• First introduced in 1998 (Khoury and Dorman)
• A discipline using genome research methods connecting epidemiology and genomic information
• Conducts population-based research
• Evaluates genomic information for population health
• Molecular epidemiology is applying molecular biology theory and technique
• Focuses on analyzing characteristics of pathogens causing particular diseases at the genetic level
• Results are used to improve medical and health care practices

HGP and Drug Design/Development

• Helped scientists understand the physiology of genetic diseases
• Aided in identifying potential genetic changes (mutations) creating the risk of a genetic disorder
• Development of strategies for drug targeting to eliminate mutations
• Genomic sequences help in determining the toxicological effect of certain compounds on an individual
• Can predict the aspects of how an individual can respond to medication by analyzing genomic makeup

HGP in Disease Discovery and Diagnosis

• Lead the field of disease discovery and diagnostics for the identification of genes
• Will reveal the inheritance pattern of monogenic diseases and other common diseases where genetics increase vulnerability
• Includes genes associated with neurodegenerative diseases, cancer, asthma, dyslexia, cystic fibrosis, diabetes, and muscular dystrophy
• The rate of progress in the field of disease discovery is presented by the rate at which the reported progress in the discovery of disease genes
• One direct clinical impact is the development of gene or DNA-based diagnostic tests

Results of HGP

• Sequenced the entire human genes
• There are approximately 30,000–35,000 genes
• Realized that any two unrelated people share ≈99.9% of their genome
• Identified single nucleotide polymorphisms (SNPs)
• Successfully drew out the human chromosome map framework